Eunpyo Moon

Atmospheric-Pressure Plasma Jet Induces Apoptosis Involving Mitochondria via Generation of Free Radicals

The plasma jet has been proposed as a novel therapeutic method for anticancer treatment. However, its biological effects and mechanism of action remain elusive. Here, we investigated its cell death effects and underlying molecular mechanisms, using air and N2 plasma jets from a micro nozzle array. Treatment with air or N2 plasma jets caused apoptotic death in human cervical cancer HeLa cells, simultaneously with depolarization of mitochondrial membrane potential. In addition, the plasma jets were able to generate reactive oxygen species (ROS), which function as surrogate apoptotic signals by targeting the mitochondrial membrane potential. Antioxidants or caspase inhibitors ameliorated the apoptotic cell death induced by the air and N2 plasma jets, suggesting that the plasma jet may generate ROS as a proapoptotic cue, thus initiating mitochondria-mediated apoptosis. Taken together, our data suggest the potential employment of plasma jets as a novel therapy for cancer.

Oncogenic potential of the DNA replication licensing protein CDT1.

The expression of a gene, designated as Retroviral insertion site (Ris)2, was activated by retroviral DNA integration in an immortalized primitive erythroid cell line, EB-PE. Ris2 was also expressed at high levels in all human tumor cell lines analysed. Consistently, NIH3T3 fibroblasts overexpressing Ris2 formed tumors in Rag2−/− mice when injected subcutaneously. The putative RIS2 protein shows a high sequence similarity to Xenopus CDT1, Drosophila DUP, and human CDT1, a newly identified DNA replication licensing protein, suggesting that Ris2 is a mouse homologue of CDT1. Cells overexpressing Ris2/Cdt1 exhibited a quicker entry into S phase when released from serum starvation compared to controls. Our results suggest that CDT1, an essential licensing protein for DNA replication, can function as an oncogene in mammals.

Targeting cancer cells with reactive oxygen and nitrogen species generated by atmospheric-pressure air plasma.

The plasma jet has been proposed as a novel therapeutic method for cancer. Anticancer activity of plasma has been reported to involve mitochondrial dysfunction. However, what constituents generated by plasma is linked to this anticancer process and its mechanism of action remain unclear. Here, we report that the therapeutic effects of air plasma result from generation of reactive oxygen/nitrogen species (ROS/RNS) including H2O2, Ox, OH−, •O2, NOx, leading to depolarization of mitochondrial membrane potential and mitochondrial ROS accumulation. Simultaneously, ROS/RNS activate c-Jun NH2-terminal kinase (JNK) and p38 kinase. As a consequence, treatment with air plasma jets induces apoptotic death in human cervical cancer HeLa cells. Pretreatment of the cells with antioxidants, JNK and p38 inhibitors, or JNK and p38 siRNA abrogates the depolarization of mitochondrial membrane potential and impairs the air plasma-induced apoptotic cell death, suggesting that the ROS/RNS generated by plasma trigger signaling pathways involving JNK and p38 and promote mitochondrial perturbation, leading to apoptosis. Therefore, administration of air plasma may be a feasible strategy to eliminate cancer cells.

Cdt1 transgenic mice develop lymphoblastic lymphoma in the absence of p53

The exact duplication of chromosomal DNA during each cell cycle ensures the correct inheritance of genetic material from mother to daughter cells. In eukaryotic cells, DNA replication can occur only when the origin of DNA replication is accurately marked by a group of proteins termed licensing proteins. One such protein is Cdt1, which is recruited first to the origin of DNA replication followed by cell division cycle 6 (Cdc6) and mini-chromosome maintenance proteins (Mcms). We previously reported that NIH3T3 cells overexpressing Cdt1 readily formed tumors in mice. To further investigate its oncogenic mechanism, we generated transgenic mice expressing Cdt1 in thymocytes. Our studies demonstrated that T-cell-directed Cdt1 transgenic mice showed normal T-cell development. However, such transgenic mice developed thymic lymphoblastic lymphoma when crossed with p53 null mice. Furthermore, tumor cells derived from NIH3T3 cells overexpressing Cdt1 displayed numerical and structural chromosomal aberrations in the form of ploidy, double minutes, translocation, inversion, chromosome end-to-end fusion and robertsonian mutation. Collectively, our studies suggest that Cdt1 overexpression most likely contributes to tumorigenecity by causing genomic instability.

Development of gusA reporter gene constructs for cereal transformation: availability of plant transformation vectors from the CAMBIA Molecular Genetic Resource Service

The use of reporter genes to characterise sequence elements that act to regulate gene expression in transgenic plants has been vital to the development of foreign gene expression strategies for use in cereal transformation. ThegusA locus ofEscherichia coli, which encodes the enzymeβ-glucuronidase (GUS), is by far the most popular reporter gene used in plant transformation. In this paper we extend the utility of the GUS reporter gene system in cereal transformation by describing and evaluating a number of novel constructs suitable for use in direct gene transfer experiments. These plasmids are all available from the Molecular Genetic Resource Service of the Center for the Application of Molecular Biology to International Agriculture.

Non-Thermal Atmospheric Pressure Plasma Inhibits Thyroid Papillary Cancer Cell Invasion via Cytoskeletal Modulation, Altered MMP-2/-9/uPA Activity

Plasma, the fourth state of matter, is defined as a partially or completely ionized gas that includes a mixture of electrons and ions. Advances in plasma physics have made it possible to use non-thermal atmospheric pressure plasma (NTP) in cancer research. However, previous studies have focused mainly on apoptotic cancer cell death mediated by NTP as a potential cancer therapy. In this study, we investigated the effect of NTP on invasion or metastasis, as well as the mechanism by which plasma induces anti-migration and anti-invasion properties in human thyroid papillary cancer cell lines (BHP10-3 and TPC1). Wound healing, pull-down, and Transwell assays demonstrated that NTP reduced cell migration and invasion. In addition, NTP induced morphological changes and cytoskeletal rearrangements, as detected by scanning electron microscopy and immunocytochemistry. We also examined matrix metalloproteinase (MMP)-2/-9 and urokinase-type plasminogen activator (uPA) activity using gelatin zymography, uPA assays and RT-PCR. FAK, Src, and paxillin expression was detected using Western blot analyses and immunocytochemistry. NTP decreased FAK, Src, and paxillin expression as well as MMP/uPA activity. In conclusion, NTP inhibited the invasion and metastasis of BHP10-3 and TPC1 cells by decreasing MMP-2/-9 and uPA activities and rearranging the cytoskeleton, which is regulated by the FAK/Src complex. These findings suggest novel actions for NTP and may aid in the development of new therapeutic strategies for locally invasive and metastatic cancers.

Non-thermal atmospheric pressure plasma induces apoptosis in oral cavity squamous cell carcinoma: Involvement of DNA-damage-triggering sub-G1 arrest via the ATM/p53 pathway

Recent advances in physics have made possible the use of non-thermal atmospheric pressure plasma (NTP) in cancer research. Although increasing evidence suggests that NTP induces death of various cancer cell types, thus offering a promising alternative treatment, the mechanism of its therapeutic effect is little understood. In this study, we report for the first time that NTP led to apoptotic cell death in oral cavity squamous cell carcinoma (OSCC). Interestingly, NTP induced a sub-G(1) arrest in p53 wild-type OSCCs, but not in p53-mutated OSCCs. In addition, NTP increased the expression levels of ATM, p53 (Ser 15, 20 and 46), p21, and cyclin D1. A comet assay, Western blotting and immunocytochemistry of γH2AX suggested that NTP-induced apoptosis and sub-G(1) arrest were associated with DNA damage and the ATM/p53 signaling pathway in SCC25 cells. Moreover, ATM knockdown using siRNA attenuated the effect of NTP on cell death, sub-G(1) arrest and related signals. Taken together, these results indicate that NTP induced apoptotic cell death in p53 wild-type OSCCs through a novel mechanism involving DNA damage and triggering of sub-G(1) arrest via the ATM/p53 pathway. These findings show the therapeutic potential of NTP in OSCC.

Combination of NTP with cetuximab inhibited invasion/migration of cetuximab-resistant OSCC cells: Involvement of NF-κB signaling.

Although the epidermal growth factor receptor (EGFR) is an established target in head-and-neck cancer (HNC), resistance to EGFR-targeted therapy mediated by various mechanisms has been reported. Therefore, a combination strategy to overcome resistance to EGFR mono-targeted therapy is clinically required. We have previously demonstrated that non-thermal atmospheric pressure plasma (NTP) induces death of various cancer cells, including oral squamous cancer (OSCC) cells. In this study, we report for the first time that combining NTP treatment with cetuximab led to inhibition of migration and invasion in cetuximab-resistant OSCC cells, which could be a promising strategy to overcome resistance to anti-EGFR therapy. NTP induced deactivation of NF-κB in SCCQLL1 cells, but not in MSKQLL1 cells. In addition, NTP increased the expression level of E-cadherin, and decreased those of vimentin, Slug, Snail, matrix metalloproteinase (MMP)-2, -9, and activities of MMPs. Moreover, NF-κB upregulation using cDNA diminished the combination effect of NTP on invasion, migration and related signals. Taken together, these results indicate that the combination of NTP with cetuximab can decrease invasiveness in cetuximab-resistant OSCCs through a novel mechanism involving the NF-κB pathway. These findings show the therapeutic potential of treatment that combines NTP and cetuximab in OSCC.

Deregulation of CREB Signaling Pathway Induced by Chronic Hyperglycemia Downregulates NeuroD Transcription

CREB mediates the transcriptional effects of glucose and incretin hormones in insulin-target cells and insulin-producing β-cells. Although the inhibition of CREB activity is known to decrease the β-cell mass, it is still unknown what factors inversely alter the CREB signaling pathway in β-cells. Here, we show that β-cell dysfunctions occurring in chronic hyperglycemia are not caused by simple inhibition of CREB activity but rather by the persistent activation of CREB due to decreases in protein phophatase PP2A. When freshly isolated rat pancreatic islets were chronically exposed to 25 mM (high) glucose, the PP2A activity was reduced with a concomitant increase in active pCREB. Brief challenges with 15 mM glucose or 30 µM forskolin after 2 hour fasting further increased the level of pCREB and consequently induced the persistent expression of ICER. The excessively produced ICER was sufficient to repress the transcription of NeuroD, insulin, and SUR1 genes. In contrast, when islets were grown in 5 mM (low) glucose, CREB was transiently activated in response to glucose or forskolin stimuli. Thus, ICER expression was transient and insufficient to repress those target genes. Importantly, overexpression of PP2A reversed the adverse effects of chronic hyperglycemia and successfully restored the transient activation of CREB and ICER. Conversely, depletion of PP2A with siRNA was sufficient to disrupt the negative feedback regulation of CREB and induce hyperglycemic phenotypes even under low glucose conditions. Our findings suggest that the failure of the negative feedback regulation of CREB is the primary cause for β-cell dysfunctions under conditions of pathogenic hyperglycemia, and PP2A can be a novel target for future therapies aiming to protect β-cells mass in the late transitional phase of non-insulin dependent type 2 diabetes (NIDDM).

Nonthermal Plasma Induces Apoptosis in ATC Cells: Involvement of JNK and p38 MAPK-Dependent ROS

Purpose To determine the effects of nonthermal plasma (NTP) induced by helium (He) alone or He plus oxygen (O2) on the generation of reactive oxygen species (ROS) and cell death in anaplastic thyroid cancer cells. Materials and Methods NTP was generated in He alone or He plus O2 blowing through a nozzle by applying a high alternating current voltage to the discharge electrodes. Optical emission spectroscopy was used to identify various excited plasma species. The apoptotic effect of NTP on the anaplastic thyroid cancer cell lines, such as HTH83, U-HTH 7, and SW1763, was verified with annexin V/propidium staining and TUNEL assay. ROS formation after NTP treatment was identified with fluorescence-activated cell sorting with DCFDA staining. The mitogen-activated protein kinase pathways and caspase cascade were investigated to evaluate the molecular mechanism involved and cellular targets of plasma. Results NTP induced significant apoptosis in all three cancer cell lines. The plasma using He and O2 generated more O2-related species, and increased apoptosis and intracellular ROS formation compared with the plasma using He alone. NTP treatment of SW1763 increased the expression of phosphor-JNK, phosphor-p38, and caspase-3, but not phosphor-ERK. Apoptosis of SW1763 as well as expressions of elevated phosphor-JNK, phosphor-p38, and caspase-3 induced by NTP were effectively inhibited by intracellular ROS scavengers. Conclusion NTP using He plus O2 induced significant apoptosis in anaplastic cancer cell lines through intracellular ROS formation. This may represent a new promising treatment modality for this highly lethal disease.